Project Summary Estrogen receptor (ER) and progesterone receptor (PR) positive breast cancers are the most prevalent subtype of the disease and constitute the majority of breast cancer-related deaths. While ER+PR+ breast tumors are initially treatable through targeted endocrine therapies, more than 1 in 3 patients will develop resistance to therapy. Moreover, ER+ breast cancers have a propensity to recur >5 years after primary tumor removal, indicative of dormant cancer stem cells (CSCs) that are responsible for drug resistance and recurrence. The role of progesterone (P) in breast cancer has been less studied than estrogens; questions remain about how progestin use in hormone replacement therapy increases breast cancer risk in postmenopausal women, and how P affects the biology of breast cancer cells. Our laboratory has identified that P directly induces a fraction of ER+PR+ breast cancer cells to become more like CSCs. This is marked by expression of several mammary stem cell markers such as cytokeratin 5 (CK5) and is accompanied by increased mammosphere formation and tumor initiation capacity, and heightened resistance to chemo- and endocrine therapies. Despite research from the 1950s showing that P modulates cellular metabolism, the influence of altered metabolic programming on the transcriptional activities of PR has not been investigated. Specifically, P suppresses oxidative phosphorylation, the primary source of reactive oxygen species within a cell. Preliminary data from our lab suggests that P modulates the glutathione antioxidant system, the most abundant scavenger of reactive oxygen and nitrogen species. This is a finding with important consequences, as breast CSCs display higher tolerance to oxidative stress. Our working hypothesis is that P suppresses oxidative phosphorylation to promote a reduced redox state permissive to CSCs with increased GSH antioxidant activity. The goal of these studies is to unravel the mechanism by which P regulates the CSC population within ER+PR+ breast cancer, and delineate the role of glutathione in toggling this switch. The specific aims of this proposal are to determine 1) P regulation of oxidative phosphorylation, 2) how P signaling alters antioxidant action, and 3) if targeting glutathione in ER+PR+ breast cancers will enhance existing therapies. These studies have potential to identify a metabolic vulnerability to exploit in targeting the CSC population in ER+PR+ breast tumors, with the ultimate goal of reducing recurrence and improving patient outcome.